1. Field of the Invention
The present invention relates to novel chemokine polypeptides and encoding nucleic acids. More specifically, therapeutic compositions and methods are provided using isolated nucleic acid molecules encoding a human chemokine beta-11 (Ck beta-11) polypeptide; and a human leukocyte adhesion inhibitor (LAI-1) polypeptide (previously termed chemokine xcex11(CKxcex11 or cka-1)), as well as Ck beta-11 and/or LAI-1 polypeptides themselves, as are vectors, host cells and recombinant methods for producing the same.
2. Related Art
Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are 8-10 kd in size. In general, chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the xe2x80x9cC-X-Cxe2x80x9d subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the xe2x80x9cCxe2x80x94Cxe2x80x9d subfamily. Thus far, at least eight different members of this family have been identified in humans.
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
Members of the xe2x80x9cC-Cxe2x80x9d branch exert their effects on the following cells: eosinophils which destroy parasites to lessen parasitic infection and cause chronic inflammation in the airways of the respiratory system; macrophages which suppress tumor formation in vertebrates; and basophils which release histamine which plays a role in allergic inflammation. However, members of one branch can exert an effect on cells which are normally responsive to the other branch of chemokines and, therefore, no precise role can be attached to the members of the branches.
While members of the Cxe2x80x94C branch act predominantly on mononuclear cells and members of the C-X-C branch act predominantly on neutrophils a distinct chemoattractant property cannot be assigned to a chemokine based on this guideline. Some chemokines from one family show characteristics of the other.
In accordance with one aspect of the present invention, there are provided novel full length or mature polypeptides which are LAI-1 and/or Ck beta-11, as well as biologically active, diagnostically useful or therapeutically useful fragments, analogs and derivatives thereof. LAI-1 and/or Ck beta-11 polypeptides or encoding nucleic acids of the present invention are preferably of animal origin, and more preferably of human origin.
In accordance with another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to Ckxcex2-11 and LAI-2 sequences.
In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides and isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
Ck beta-11 Polynucleotides. The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the Ck beta-11 polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 75948 on Nov. 11, 1994. The nucleotide sequence determined by sequencing the deposited Ck beta-11 clone, which is shown in FIG. 1 (SEQ ID NO: 1), contains an open reading frame encoding a polypeptide of 98 amino acid residues, with a leader sequence of about 17 amino acid residues, and a predicted molecular weight for the mature protein of about 10 kDa in non-glycosylated form, and about 10-14 kDa in glycosylated form, depending on the extent of glycoslyation. The amino acid sequence of the mature Ck beta-11 protein is shown in FIG. 1, as amino acid residues 18-98 of SEQ ID NO:2.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (1)(a) a nucleotide sequence encoding an Ck beta-11 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2); (1)(b) a nucleotide sequence encoding the mature Ck beta-11 polypeptide having the amino acid sequence at positions 18-98 in FIG. 1 (SEQ ID NO:2); (1)(c) a nucleotide sequence encoding the Ck beta-11 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75948; (1)(d) a nucleotide sequence encoding the mature Ck beta-11 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75948; and (1)(e) a nucleotide sequence complementary to any of the nucleotide sequences in (1)-(a), (b), (c) or (d) above.
LAI-1 Polynucleotides. In one aspect, the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the LAI-1 polypeptide having the amino acid sequence shown in FIG. 2 (SEQ ID NO:4) or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 75947 on Nov. 11, 1994. The nucleotide sequence determined by sequencing the deposited LAI-1 clone, which is shown in FIG. 2 (SEQ ID NO:3), contains an open reading frame encoding a polypeptide of 109 amino acid residues, with a leader sequence of about 22 amino acid residues, and a predicted molecular weight of about 110 kDa in non-glycosylated form, and about 11-14 kDa in glycosylated form, depending on the extent of glycoslyation. The amino acid sequence of the mature LAI-1 protein is shown in FIG. 2, as amino acid residues 23-109 of SEQ ID NO:4.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (2)(a) a nucleotide sequence encoding the LAI-1 polypeptide having the complete amino acid sequence in FIG. 2 (SEQ ID NO:4); (2)(b) a nucleotide sequence encoding the mature LAI-1 polypeptide having the amino acid sequence at positions 23-109 in FIG. 2 (SEQ ID NO:4); (2)(c) a nucleotide sequence encoding the LAI-1 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75947; (2)(d) a nucleotide sequence encoding the mature LAI-1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.75947; and (2)(e) a nucleotide sequence complementary to any of the nucleotide sequences in (2)-(a), (b), (c) or (d) above.
Ck beta-11 and LAI-1 Polynucleotide Variants. The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of FIGS. 1 and 2 (SEQ ID NOS:2 and 4) or the polypeptides encoded by the cDNA of the deposited clone(s). The variants of the polynucleotides can be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.
Homologous Ck beta-11 and LAI-1 Polynucleotides. Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% homologous or identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (1)-, (2)- or (3)-(a), (b), (c), (d) or (e), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (1)- or (2)-(a), (b), (c), (d) or (e), above. These polynucleotides which hybridize do not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
Nucleic Acid Probes. In accordance with yet another aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the Ck beta-11 and/or LAI-1 nucleic acid sequences.
Recombinant Vectors, Host Cells and Expression. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of Ck beta-11 and/or LAI-1 polypeptides or peptides by recombinant techniques.
Ck beta-11 Polypeptides. The invention further provides an isolated Ck beta-11 polypeptide having an amino acid sequence selected from the group consisting of: (1)(a) the amino acid sequence of the Ck beta-11 polypeptide having the complete 98 amino acid sequence, including the leader sequence shown in FIG. 1 (SEQ ID NO:2); (1)(b) the amino acid sequence of the mature Ck beta-11 polypeptide (without the leader) having the amino acid sequence at positions 18-98 in FIG. 1 (SEQ ID NO:2); (I)(c) the amino acid sequence of the Ck beta-11 polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 75948; and (I)(d) the amino acid sequence of the mature Ck beta-11 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.75948.
LAI-1 Polypeptides. The invention further provides an isolated LAI-1 polypeptide having an amino acid sequence selected from the group consisting of: (II)(a) the amino acid sequence of the LAI-1 polypeptide having the complete 109 amino acid sequence, including the leader sequence shown in FIG. 2 (SEQ ID NO:4); (II)(b) the amino acid sequence of the mature LAI-1 polypeptide (without the leader) having the amino acid sequence at positions 23-109 in FIG. 2 (SEQ ID NO:4); (II)(c) the amino acid sequence of the LAI-1 polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 75947; and (II)(d) the amino acid sequence of the mature LAI-1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75947.
Homologous Ck beta-11 and/or LAI-1 Polypeptides. Polypeptides of the present invention also include homologous polypeptides having an amino acid sequence with at least 90% identity, and more preferably at least 95% identity to those described in (I)- and (II)-(a), (b), (c) or (d) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
Ck beta-11 and/or LAI-1 Epitope Bearing Polypeptides and Encoding Polynucleotides. An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of an Ck beta-11 and/or LAI-1 polypeptide having an amino acid sequence described in (I)- or (II)-(a), (b), (c) or (d), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of an Ck beta-11 and/or LAI-1 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of an Ck beta-11 and/or LAI-1 polypeptide having an amino acid sequence in (I)- or (II)-(a), (b), (c) or (d), above.
Ck beta-11 and/or LAI-1 Antibodies. In accordance with yet a further aspect of the present invention, there is provided an antibody against such polypeptides. In another embodiment, the invention provides an isolated antibody that binds specifically to an Ck beta-11 and/or LAI-1 polypeptide having an amino acid sequence described in (I)- and/or (II)-(a), (b), (c) or (d) above.
The invention further provides methods for isolating antibodies that bind specifically to an Ck beta-11 and/or LAI-1 polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
Ck beta-11 and/or LAI-1 Polypeptides, Agonists, Antagonists and Methods. In accordance with yet another aspect of the present invention, there are provided polypeptides, agonists, antagonists or inhibitors of such polypeptides, which can be used to modulate the action of such polypeptides, agonists, antagonists or inhibitors, e.g., in the treatment of arteriosclerosis, autoimmune and chronic inflammatory and infective diseases, histamine-mediated allergic reactions, hyper-eosinophilic syndrome, silicosis, sarcoidosis, inflammatory diseases of the lung, inhibition of IL-1 and TNF, aplastic anaemia, and myelodysplastic syndrome. Alternatively, such polypeptides can be used to inhibit production of IL-1 and TNF-xcex1 in any related diseases, e.g., to treat aplastic anemia, myelodysplastic syndrome, inflammatory diseases, diabetes, asthma and arthritis.
Such polypeptides, agonists, antagonists or inhibitors, can also be used to modulate (e.g., enhance or inhibit) the action of such polypeptides, for example, in the treatment of certain autoimmune diseases, atherosclerosis, chronic inflammatory and infectious diseases, histamine and IgE-mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, cancers, silicosis, sarcoidosis, rheumatoid arthritis, shock, hyper-eosinophilic syndrome and fibrosis in the asthmatic lung.
Diagnostic Assays. In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to the underexpression and overexpression of the polypeptides and for detecting mutations in the nucleic acid sequences encoding such polypeptides.
In accordance with yet another aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, as research reagents for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors, for the purpose of developing therapeutics and diagnostics for the treatment of human disease.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by an Ck beta-11 and/or LAI-1 polypeptide, which involves contacting cells which express the Ck beta-11 and/or LAI-1 polypeptide with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
For a number of disorders, it is believed that significantly higher or lower levels of Ck beta-11 and/or LAI-1 gene expression can be detected in certain tissues or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a xe2x80x9cstandardxe2x80x9d Ck beta-11 and/or LAI-1 gene expression level, ie., the Ck beta-11 and/or LAI-1 expression level in tissue or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves: (a) assaying Ck beta-11 and/or LAI-1 gene expression level in cells or body fluid of an individual; (b) comparing the Ck beta-11 and/or LAI-1 gene expression level with a standard Ck beta-11 and/or LAI-1 gene expression level, whereby an increase or decrease in the assayed Ck beta-11 and/or LAI-1 gene expression level compared to the standard expression level is indicative of a disorder. Such disorders include leukemia, chronic inflammation, autoimmune diseases, solid tumors.
Pharmaceutical Compositions. The present invention also provides, in another aspect, pharmaceutical compositions comprising at least one of an Ck beta-11 and/or LAI-1: polynucleotide, probe, vector, host cell, polypeptide, fragment, variant, derivative, epitope bearing portion, antibody, antagonist, agonist.
Therapeutic Methods. In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, for example, to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy, to remove leukemic cells, to stimulate an immune response, to regulate hematopoiesis and lymphocyte trafficking, treatment of psoriasis, solid tumors, to enhance host defenses against resistant and acute and chronic infection, and to stimulate wound healing.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, agonists, antagonists, or inhibitors, or polynucleotides encoding such polypeptides, for therapeutic purposes, for example, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-immune diseases, parasitic infections, psoriasis, asthma, allergy, to regulate hematopoiesis, to stimulate growth factor activity, to inhibit angiogenesis and to promote wound healing.
Alternatively or additionally, such treatment includes arteriosclerosis, autoimmune and chronic inflammatory and infective diseases, histamine-mediated allergic reactions, hyper-eosinophilic syndrome, silicosis, sarcoidosis, inflammatory diseases of the lung, inhibition of IL-1 and TNF, aplastic anaemia, and myelodysplastic syndrome. Alternatively, such polypeptides can be used to inhibit production of IL-1 and TNF-xcex1 in any related diseases, e.g., to treat aplastic anemia, myelodysplastic syndrome, inflammatory diseases, diabetes, asthma and arthritis.
Such polypeptides, agonists, antagonists or inhibitors, can also be used to treat (e.g., enhance or inhibit) the action of such polypeptides, agonists, antagonists or inhibitors, e.g, in the treatment of certain autoimmune diseases, atherosclerosis, chronic inflammatory and infectious diseases, histamine and IgE-mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, cancers, silicosis, sarcoidosis, rheumatoid arthritis, shock, hyper-eosinophilic syndrome and fibrosis in the asthmatic lung.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of Ck beta-11 and/or LAI-1 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated Ck beta-11 and/or LAI-1 polypeptide, agonist, antagonist or inhibitor, of the invention.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of Ck beta-11 and/or LAI-1 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an Ck beta-11 and/or LAI-1 polypeptide, agonist, antagonist or inhibitor, of the invention.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.